Summary

Using conditional gene targeting in mice, we show that the chromatin
remodeler Mi-2β is crucial for different aspects of skin development.
Early (E10.5) depletion of Mi-2β in the developing ventral epidermis
results in the delayed reduction of its suprabasal layers in late
embryogenesis and to the ultimate depletion of its basal layer. Later (E13.5)
loss of Mi-2β in the dorsal epidermis does not interfere with suprabasal
layer differentiation or maintenance of the basal layer, but induction of hair
follicles is blocked. After initiation of the follicle, some subsequent
morphogenesis of the hair peg may proceed in the absence of Mi-2β, but
production of the progenitors that give rise to the inner layers of the hair
follicle and hair shaft is impaired. These results suggest that the extended
self-renewal capacity of epidermal precursors arises early during
embryogenesis by a process that is critically dependent on Mi-2β. Once
this process is complete, Mi-2β is apparently dispensable for the
maintenance of established repopulating epidermal stem cells and for the
differentiation of their progeny into interfollicular epidermis for the
remainder of gestation. Mi-2β is however essential for the reprogramming
of basal cells to the follicular and, subsequently, hair matrix fates.

INTRODUCTION

The stratified epithelium of the epidermis provides the barrier between the
organism and the outside world (Fuchs and
Raghavan, 2002; Watt,
1998). The cells of the basal layer of the epidermis are
comparatively undifferentiated and proliferate to generate progeny that detach
from the basement membrane and progressively differentiate as they are
displaced through the successive layers of the epidermis before being shed
from the surface. Homeostasis of the mature epidermis is thought to depend on
the cycling of stem cells in the basal layer, although it remains unclear
whether there are dedicated stem cells that self renew while giving rise to
more committed `transient amplifying' (TA) basal cells, or whether basal cells
are more generally equipotent. The differentiation of the epidermis of the
mouse begins at E8.5, as the embryonic ectoderm begins to express epithelial
markers such as the nuclear factor p63 (also known as Trp63 - Mouse Genome
Informatics) and keratins 8 and 18
(Jackson et al., 1981;
Koster et al., 2004).
Ectodermal commitment to the epidermal lineage occurs between E9.5 and E12.5,
producing a basal layer expressing keratins 5 and 14 and a second, transitory
layer known as the periderm (Byrne et al.,
2003). Further differentiation commences at E14.5, as the
intermediary layer and then the spinous and granular layers of the mature
epidermis are formed. The final stages of differentiation include the
formation of a cornified layer and the acquisition of barrier function, events
that occur just prior to birth. This progression of development in embryonic
epidermis is demarcated by the expression of structural proteins in the newly
formed layers, a pattern that is recapitulated by individual keratinocytes as
they transit from the basement membrane to the surface in mature
epidermis.

During skin development, a subset of keratinocytes is recruited to form
hair follicles. This fate decision is guided by inductive interactions with an
underlying population of mesenchymal cells, some of which eventually form the
dermal papilla (DP) (Hardy,
1992). These epithelial-mesenchymal interactions lead to the
downgrowth of a hair peg and to the formation of a hair bulb, in which
keratinocytes proliferate and differentiate into distinct concentric layers of
epithelial cells that constitute the inner root sheath and the hair shaft
(Sengel and Mauger, 1976). In
the mouse, pelage hairs consist of different types of hair follicles that are
formed in successive waves during embryogenesis
(Hardy, 1992). Primary
(tylotricht) follicles initiate development at E14.5 and are characterized by
two sebaceous glands and a large hair bulb that gives rise to a long straight
hair. Induction of secondary hair follicles that produce awls hairs begins at
E16.5. A final wave of follicle formation in late gestation and after birth
gives rise to the zigzag and auchene hairs.

Gene expression changes associated with the partially characterized genetic
hierarchy that guides follicle development serve as markers of specific steps
in follicle development (Millar,
2002). Activation of the canonical Wnt/β-catenin pathway is
required for the initial formation of hair placodes in all three waves
(Andl et al., 2002). Another
early step in placode development is a local increase in the expression of the
ectodysplasin-A receptor (Edar), which is expressed at low levels throughout
the basal epidermis before placode formation. The local increase in Edar
expression is followed rapidly by a decrease in E-cadherin (cadherin 1)
expression and induction of both sonic hedgehog (Shh) and P-cadherin (cadherin
3) expression in the epithelial cells in contact with the forming DP
(Hardy and Vielkind, 1996;
Headon and Overbeek, 1999;
Jamora et al., 2003).
Expression of bone morphogenetic proteins 2 and 4 (Bmp2 and Bmp4) in the
mesenchyme indicates the formation of the DP, and subsequent Wnt5a expression
in the DP reflects its further maturation that is dependent on Shh expression
in the epidermal placode (Reddy et al.,
2001; Wilson et al.,
1999). Although the disruption of components of these signaling
pathways might have preferential effects on specific waves of follicle
formation, this sequence of gene expression is shared by follicles in all
three waves.

Commitment to the epidermal lineage and subsequent decisions between
interfollicular and follicular cell fates rely on a balance between positive
and negative events in gene expression. Sequence-specific transcription
regulators have been implicated in lineage decisions and function in part by
targeting genes whose expression supports lineage progression. p63, a member
of the p53 family of DNA-binding factors, is a key regulator of epidermal
differentiation as its ectopic expression in simple epithelia induces
expression of epidermal keratins and presumably induces the squamous cell fate
(Koster et al., 2004). The
abilities of lineage-determining DNA-binding factors to either access their
chromosomal sites and/or provide permanence to the regulation of the
associated locus is central to lineage commitment. Chromatin regulators
function in concert with lineage-specific factors to provide long-term
epigenetic regulation (Kim et al.,
1999). These include ATP-dependent remodelers, histone
deacetylases (HDACs), histone acetyltransferases (HATs) and methylases, which
are enzymes that can transiently or permanently change the accessibility of
genes to transcriptional machineries. Chromatin regulators can generate
epigenetic markings on chromatin that underlie the cell's memory and allow for
the stable propagation of lineage-specific expression profiles through
multiple divisions during development
(Georgopoulos, 2002).

Mi-2α and Mi-2β (also known as Chd3 and Chd4, respectively -
Mouse Genome Informatics) are closely related genes encoding ATP-dependent
chromatin remodelers (Seelig et al.,
1996). Mi-2β is expressed at significantly higher levels than
Mi-2α in developing and adult tissues and is observed in the skin,
mucosal epithelia, the thymus, the kidney, specific areas of the brain, and in
the hemopoietic foci of the liver of the mouse embryo
(Kim et al., 1999). Mi-2
proteins contain two PHD (plant homeodomain) zinc-finger domains, two chromo
domains and a SWI2/SNF2-type helicase/ATPase domain. They reside in the
nucleosome remodeling histone deacetylase (NURD) complex that includes the
histone deacetylases Hdac1 and Hdac2, two histone-binding proteins RbAp46 and
RbAp48 (also known as Rbbp7 and Rbbp4, respectively - Mouse Genome
Informatics), and the metastasis-associated proteins Mta1 and Mta2
(Hassig et al., 1998;
Xue et al., 1998;
Zhang et al., 1998). Because
of the Mi-2 association with HDAC, it was thought to be involved primarily in
establishing a repressive chromatin environment by cooperating with the HDACs
of the NURD complex. However, recent reports indicate that Mi-2 can
participate in other regulatory activities that relate to transcriptional
elongation, termination (Alen et al.,
2002; Krogan et al.,
2003), chromatid cohesion
(Hakimi et al., 2002), and
positive regulation of gene expression
(Hirose et al., 2002;
Williams et al., 2004).

In the immune system, much of the Mi-2β is found in a stable complex
with members of the Ikaros family of lymphoid-lineage-determining factors and
HDACs in the NURD complex (Kim et al.,
1999; O'Neill et al.,
2000). However, during T-cell development, Mi-2β also acts in
association with the E-box DNA-binding protein HEB (also known as Tcf12 -
Mouse Genome Informatics) and the HAT p300 as a positive regulator of the
Cd4 gene, a hallmark in the differentiation of the helper T-cell
lineage. Conditional inactivation of Mi-2β during T-cell development
revealed that it is required to generate a chromatin environment at
positive-acting regulatory elements that is conducive to Cd4 expression,
thereby setting the stage for lineage-specific transcription factors to drive
this developmental decision in the T lineage. These studies also revealed a
key role for Mi-2β in the transition from the double negative to the
double positive stage of thymocyte differentiation, and in mature T cells in
promoting antigen-mediated proliferative responses
(Williams et al., 2004).

The high levels of Mi-2β expression in the embryonic ectoderm and its
preferential expression in the hair placode and the matrix of the hair
follicle (Fig. 1A) prompted us
to examine the role of Mi-2β in skin development. The conditional allele
of Mi-2β was inactivated in keratinocytes at distinct stages of epidermal
development and new insights into the regulatory events that control
development and homeostasis of the epidermis and its appendages were
revealed.

MATERIALS AND METHODS

Mice

The generation and characterization of the
Mi-2βLoxPF/LoxPF mice have been described previously
(Williams et al., 2004).
K14-Cre transgenic mice obtained from Dr P. Chambon
(Li et al., 2001) were mated
to Mi-2βLoxPF/LoxPF mice to generate mice homozygous
for loss of Mi-2β function in the epidermis. PCR genotyping was performed
using the genomic DNA isolated from P1 dorsal epidermis. Briefly, epidermis
was separated from dermis by treating with 0.05% collagenase overnight at
4°C. PCR primers are as follows:

Cycling conditions for all the reactions were as follows: 35 cycles of 30
seconds at 94°C, 1 minute at 56°C and 1 minute at 72°C.

Histology

For histopathology, tissue samples were frozen in OCT or fixed in 3.7%
formalin and embedded in paraffin. Sections (4 μm) were stained with
Hematoxylin and Eosin. For immunofluorescence, TUNEL, and in situ
hybridization, tissue samples were frozen in OCT and sectioned at 6 μm.

TUNEL analysis

TUNEL was performed according to the manufacturer's protocol (Promega).
Briefly, sections were fixed in 3.7% formalin, permeabilized by proteinase K
digestion, and subjected to a TdT reaction. The TdT label was detected by
DAB.

Expression of Mi-2β mRNA during epidermal development and
conditional inactivation of Mi-2β in the skin. (A) In
situ hybridization studies reveal Mi-2β mRNA expression at E10.5, E14.5,
E18.5 and P1. Mi-2β is uniformly expressed in the embryonic ectoderm
(E10.5). Mi-2β transcripts are also detected in E14.5 epidermis in the
basal and stratum intermediate layers. In the differentiating hair follicle,
increased levels of Mi-2β mRNA are first detected in the placode
(arrowhead) and then in the matrix (arrow). Scale bar: 50 μm. (B)
Cre-dependent conversion of the floxed allele (LoxPF) to the mutant allele
(ΔF) in the P1 dorsal epidermis revealed by PCR of genomic DNA (lanes 2
and 4). (C) Wild-type (WT) and mutant littermates at P1. (D)
Hematoxylin and Eosin-stained cross-sections of WT and mutant skin at P1. The
dotted line demarcates the dorsal region and the unbroken line the ventral
region. The mutant skin exhibits an exacerbation of phenotypes from the dorsal
to the ventral side that includes thinning of the epidermis and reduction in
hair follicles. Scale bar: 100 μm. A further magnification of the mutant
skin is provided beneath to show the absence of the basal layer and the
thinning of the suprabasal and cornified layers in the ventral region (right),
and the more normal epidermal differentiation in the dorsal region (left).
(E) The presence of Mi-2β protein (green) in WT (left) and in the
dorsal (middle) and ventral (right) mutant skin was evaluated by
immunofluoresence at successive stages of development. DAPI-stained nuclei are
shown in red, and the white dotted line demarcates the dermal-epidermal
junction. Expression of Mi-2β protein is indicated by the presence of
yellow nuclei, depletion of Mi-2β by red nuclei. Depletion of Mi-2β
protein occurs earlier (E10.5) in the ventral skin, and later (E13.5 and
later) in the dorsal skin. Scale bar: 50 μm.

Quantitative histomorphometry

The number of hair follicles per unit length of epidermis was counted in
frozen and paraffin sections of Mi-2β mutant dorsal skin (n=5)
at E18.5 and P1, and was compared with that of age-matched wild-type (WT) skin
(n=3). The percentage of hair follicles at different stages of
morphogenesis was assessed. These stages were defined on the basis of accepted
morphological criteria (Hardy,
1992). At least 151 longitudinal hair follicles in 63 microscopic
fields derived from five Mi-2β mutant animals were compared with those of
290 hair follicles from five age-matched WT mice at E18.5. At least 412
longitudinal hair follicles in 66 microscopic fields derived from five
Mi-2β mutant animals were compared with those of 436 hair follicles from
three age-matched WT mice at P1.

RT-PCR

RNA was extracted from P1 dorsal epidermis with Trizol (Invitrogen)
according to the manufacturer's protocol. cDNA was generated using random
primers and the Superscript II Kit (Invitrogen). The cDNA was amplified by PCR
using the following conditions: 28-35 cycles of 30 seconds at 94°C, 45
seconds at 57°C and 45 seconds at 72°C. The PCR products were ligated
into the pCRII TA vector (Invitrogen) and verified by sequencing. The primers
used for PCR were:

RESULTS

Mi-2β expression during epidermal differentiation

To gain insight into the role of Mi-2β during epidermal morphogenesis,
we examined the pattern of Mi-2β mRNA expression during development. At
E10.5, the embryonic ectoderm consists of a single-cell layer in which
Mi-2β was uniformly and highly expressed
(Fig. 1A, E10.5). At E14.5, the
ectodermal layer had begun to differentiate into the basal and stratum
intermediate layers, which express Mi-2β
(Fig. 1A, E14.5). At this stage
of development, hair placodes expressing Mi-2β were also observed.
Increased levels of Mi-2β mRNA were detected in the growing tip of the
hair peg and persisted in the cells that form the matrix of the
differentiating hair follicle (Fig.
1A, E14.5, E18.5 and P1). In contrast to the distinctive pattern
of expression in the hair follicle, Mi-2β mRNA was expressed at
relatively low levels in the mature interfollicular epidermis
(Fig. 1A, E18.5 and P1).

Mi-2β inactivation in the epidermis causes abnormal development
of the integument

The role of Mi-2β during development of the epidermis and hair was
evaluated using a conditional inactivation strategy. Mice containing an
Mi-2β allele with loxP sites flanking the ATPase domain were crossed to a
K14cre transgenic line that expresses cre recombinase in the basal
epidermis and the outer root sheath of the hair follicle
(Li et al., 2001). As
previously shown (Williams et al.,
2004), cre recombinase removes sequences encoding the ATPase
domain required for Mi-2β remodeling activity, resulting in a mutant mRNA
that does not produce stable protein. As revealed by genomic PCR, the floxed
Mi-2β alleles (Mi-2βloxPF/loxpF) were efficiently
disrupted by K14cre in the epidermis (Fig.
1B).

Mice homozygous for the Mi-2βLoxPF allele
and carrying the K14cre transgene die within 24 hours of birth. The
skin of these mutant mice is shiny and flaky
(Fig. 1C), exhibits a severe
reduction in hair follicles and shows abnormal whisker hairs (data not shown).
A curly tail is another morphological phenotype of the K14cre
deletion of Mi-2β (Fig.
1C). Histological analysis of the mutant skin demonstrated a
marked difference in phenotypes between the dorsal and ventral areas
(Fig. 1D). In the dorsolateral
region, a relatively normal multilayered epidermis was detected, but the
number and length of hair follicles were drastically reduced. In most of the
ventral region, the structure of the epidermis was greatly affected, with most
layers reduced in size. Hair follicles were also absent. A stratum corneum was
present in both areas (Fig.
1D). Occasional patches of multilayered skin lacking hair
follicles were observed among the severely depleted ventral skin of the mutant
(see Fig. S1 in the supplementary material).

The difference in phenotype between the dorsal and ventral skin could be
explained either by a fundamental difference in the requirement for Mi-2β
in the development of dorsal and ventral skin, or by a difference in the
timing of Mi-2β protein depletion. Examination of Mi-2β protein at
E10.5 revealed its dramatic depletion in most of the ventral skin, while
expression persisted in dorsal skin (Fig.
1E). Mi-2β was still detectable at E13.5 in dorsal epidermis
(Fig. 1E). However, by E14.5,
extensive depletion of Mi-2β protein was also observed in dorsal skin,
although some areas of persistent expression were apparent at this and later
stages of development (Fig. 1E
and data not shown). Thus, the more severe phenotype in the ventral region of
the skin is associated with depletion of Mi-2β during the earliest stages
of epidermal development, whereas the distinct phenotypes seen in dorsal skin
occur when Mi-2β is removed after the initiation of epidermal
differentiation.

Effects of Mi-2β depletion prior to epidermal
differentiation

Although Mi-2β depletion in the ventral epidermis occurs at E10.5,
ventral skin from embryos at E14.5 was histologically normal
(Fig. 2A, mutant E14.5). The
effects of Mi-2β depletion were not detected until E16.5, when expansion
and maturation of the stratified layers became apparent
(Fig. 2A, mutant E16.5). At
this and subsequent stages, the epidermis was markedly reduced in thickness
and no appendages were detected (Fig.
2A, mutant E16.5-P1). A thin, cornified layer could be discerned
in ventral skin even in the most severely affected areas
(Fig. 1D;
Fig. 2A, mutant P1).

Keratin 5 (K5), keratin 1 (K1) and loricrin serve as markers of the basal,
suprabasal and granular layers, respectively, in mature skin and are also
indicative of the formation of these layers during embryonic development. K5
was expressed in a wild-type (WT) pattern in the innermost layer of the mutant
epidermis up to E14.5-16.5 (Fig.
2B). However, after E16.5, a progressive reduction in the
K5-expressing layer was seen. Strikingly, by P1, some areas of the skin failed
to express any K5 (Fig. 2B,
mutant P1-2). K1 expression in the suprabasal layer of WT skin was readily
detected by E13.5 (Fig. 2C,
WT). In the mutant skin, induction of K1 expression was delayed to E14.5
(Fig. 2C, mutant). A
progressive reduction in the K1-expressing layer was seen from E16.5 to P1,
with some areas completely lacking the suprabasal layer
(Fig. 2C, mutant P1-2).
Finally, loricrin, which demarcates the granular layer, is normally detected
by E14.5 and its induction was not influenced by the absence of Mi-2β
(Fig. 2D, mutant). Nonetheless,
by P1, the loricrin-expressing layer was greatly reduced in the mutant skin
with very little staining detected (Fig.
2D, mutant).

Thus, Mi-2β depletion at an early stage of development, prior to or
during ectodermal commitment to the epidermal lineage, severely affects
epidermal differentiation. The initially normal induction and expression of
squamous-cell-layer markers indicates that there is no major defect in the
differentiation of the cell types of the epidermis. However, the progressive
depletion of the lower layers of the skin during development suggests either a
defect in the ability of epidermal stem cells to renew themselves or to
continue to generate their more differentiated progeny. Defects in
proliferation, survival or differentiation of the cells of the basal layer of
the epidermis could give rise to this general thinning of the epidermis and
depletion of the lower layers. However, at both E14.5 and E16.5, a similar
number of Pcna-positive cells were detected in the basal layer of the ventral
mutant as compared with WT skin (Fig.
3A,B), indicating that there is no initial defect in the
proliferation of the basal cells. By contrast, from E18.5 through P1, a
significant reduction of Pcna-positive cells was seen in the basal layer of
ventral mutant skin (Fig.
3A,B). A possible effect of Mi-2β depletion on apoptosis
during epidermal differentiation was also examined. A process akin to
apoptotic cell death normally occurs during terminal differentiation in the
uppermost layer of the WT skin, but little or no apoptosis is normally
observed in the basal layer (Fig.
3C, WT ventral). In the mutant skin, the persistence of nuclei in
the uppermost layers of the skin accounts for the increased number of
TUNEL-positive cells observed (Fig.
3C, mutant ventral). However, no increase in apoptosis was
observed in the basal or immediate suprabasal layers of many of those areas
where the depletion of Mi-2β lead to a dramatic thinning of the
epidermis. Thus, the abnormal differentiation of the skin can be attributed in
part to a defect in renewal of the basal epidermis, rather than to the death
of this cell population.

. Early depletion of Mi-2β in the ventral epidermis results
in late depletion of the basal and suprabasal layers. (A)
Hematoxylin and Eosin-stained sections of WT and mutant ventral skin isolated
from E10.5-P1 stages of development. A reduced cellularity in the ventral
epidermal layers was apparent from E16.5 to P1. (B-D) Expression of
keratin 5 (K5) (basal epidermis), keratin 1 (K1) (suprabasal epidermis), and
loricrin (granular epidermis) was examined by immunofluoresence. DAPI-stained
nuclei are shown in blue. The timing of induction and expression of these
epidermal differentiation markers is not initially affected. Their progressive
reduction later in development reflects a progressive depletion of basal and
suprabasal layers. Scale bars: 50 μm.

Effects of Mi-2β depletion during epidermal differentiation

Mi-2β depletion occurred later in development in the dorsal epidermis,
beginning at E13.5 during the onset of epidermal differentiation
(Fig. 1E). In contrast to
ventral skin, the dorsal mutant epidermis appeared relatively normal and
multilayered throughout development, although the number and length of hair
follicles was drastically reduced (Fig.
1D). With the exception of a modest delay in the onset of K1
expression (Fig. 4B, E13.5 and
E14.5), the initial appearance and subsequent maturation of the basal,
intermediate and granular layers of the epidermis were largely
indistinguishable from WT. Even in regions where Mi-2β had been deleted
throughout the epidermis, K5, K1 and loricrin staining were similar to that in
WT skin (Fig. 4A-C).

. Late effects on proliferation and apoptosis in the
Mi-2β-depleted ventral epidermis. (A,B) The
percent of Pcna-positive cells within the basal layer (K5-positive) of WT and
mutant skin was estimated during development. Sagittal sections were
co-labeled with antibodies against the proliferation marker Pcna and K5.
Nuclei were counterstained with DAPI (A). The data (B) represent the mean
percentage of Pcna-positive cells within the basal layer (K5- and
DAPI-positive) from five independent animals. From E14.5 through E16.5, a
similar number of Pcna-positive basal cells were seen in both the mutant and
WT skin. However, starting at E18.5 and through P1, a significant reduction of
Pcna-positive cells was detected in the ventral, but not the dorsal, mutant
regions. (C) Apoptotic cell nuclei (brown) were detected by TUNEL
analysis on ventral skin at P1. In both the WT and mutant, TUNEL-positive
cells were detected in the uppermost layer of the epidermis, but the dramatic
increase in their number seen in the mutant suggests persistence of nucleated
dead cells and a defect in terminal differentiation. Scale bars: 50 μm.

A notable defect in the dorsal epidermis is the expression of keratin 6
(K6). K6 is expressed in the periderm that overlies the developing epidermis
(McGowan and Coulombe, 1998).
After the cornified layer has fully developed around E17.5, the periderm
sloughs off and K6 expression is confined to the hair follicle. In both the WT
and Mi-2β-depleted skin, a K6-expressing periderm was detected at E16.5
and was shed by E18.5 (Fig. 4D,
E16.5 and E18.5). In sharp contrast to the situation in the WT, cells in the
suprabasal layers that lacked Mi-2β expressed K6 starting at E18.5
(Fig. 4D, mutant). A barrier
defect could explain the K6 induction in the suprabasal layers
(Paladini et al., 1996).
However, the barrier was largely intact on the dorsal side
(Fig. 4E). Analysis of regions
where mosaic depletion of Mi-2β had occurred demonstrated that K6
expression was confined to cells that lacked Mi-2β
(Fig. 4F). Mi-2β-deficient
cells interspersed among WT cells specifically expressed K6, whereas cells
that retained Mi-2β in largely deleted areas lacked K6 despite its
expression in immediately adjacent cells. Furthermore, aberrant K6 expression
was confined to the suprabasal layers. Although the induction of K6 might
depend on a signal induced in response to a subtle defect in epidermal
structure or barrier function (Fig.
4E), this signal is only adequate to induce K6 in suprabasal cells
that lack Mi-2β.

Mosaic Mi-2β depletion severely affects hair follicle
morphogenesis

Although differentiation of the dorsal interfollicular epidermis was for
the most part intact in the mutants, the development of hair follicles was
severely compromised in the absence of Mi-2β. Hair follicle induction
normally occurs during the period that Mi-2β is being deleted in a mosaic
fashion in the dorsal epidermis. Despite the variable nature of this deletion
pattern, dramatic differences in hair follicle development were evident when
whole skin was evaluated (Fig.
5). Such effects on hair follicle development were even more
dramatic when the analysis was confined to areas where Mi-2β depletion
was complete.

Effects of Mi-2β depletion in the dorsal epidermis.
(A-D) Development of the basal and suprabasal layers was examined by
immunofluoresence using antibodies to K5, K1, loricrin and keratin 6 (K6).
DAPI-stained nuclei are shown in blue. Expression of K5, K1 and loricrin was
similar in WT and mutant throughout development (A-C). The K6-positive
periderm detected at E16.5 was shed at E18.5 in both WT and mutant (D). From
E18.5 through P1, K6 induction was observed in the suprabasal layers of the
mutant but not WT skin (D). (E) Skin barrier function was analyzed by a
barrier-dependent dye exclusion assay at E19.5. The WT and, for the most part,
the mutant dorsal epidermis prevented dye penetration indicating intact
barrier function. By contrast, the ventral part of the mutant epidermis was
readily penetrated by the dye indicating lack of a barrier. (F) K6
expression (red) is confined to cells that lack Mi-2β (green). Scale bar:
50 μm.

At E18.5, a later wave of hair follicle morphogenesis is occurring and
tylotrich follicles have reached stage 3-4, while awl follicles have reached
stage 1-2 in the WT (Fig. 5A).
At this time, only half the number of hair follicles seen in the WT were
detected in the mutant (Fig.
5A,B). Furthermore, hair follicles at advanced stages of
morphogenesis were greatly reduced: about 20% of hair follicles in the WT had
reached stage 3b and above, whereas only 5% were this mature in the mutant
(Fig. 5C, E18.5). By P1, the
total number of follicles in the mutant was still 50% of WT, and the
distribution of developmental stages remained distinct
(Fig. 5B,C, P1). In the WT,
many tylotrich follicles and awl follicles had reached stage 4-6. In addition,
newly forming hair follicles at stage 1-2 were readily detected
(Fig. 5A,C, P1 WT). By
contrast, in the mutant, follicles at stage 1 and at stages 4-6 (stage 4 and
above) were greatly reduced or even absent. The majority of the follicles
(79%) were distributed between stages 2 and 3b, whereas only 45% of the
follicles were in this range in WT skin
(Fig. 5A,C, P1). This distinct
developmental stage distribution of hair follicles in the mutant versus the WT
epidermis is suggestive of discrete defects occurring during follicle
initiation and during the later stages of follicular morphogenesis, rather
than of a general delay in appendage development.

. Mi-2β depletion causes severe effects on hair follicle
morphogenesis. (A) Hematoxylin and Eosin-stained sagittal sections
of dorsal skin from WT and mutant at E18.5 and P1. The number next to the
follicles designates their developmental stage according to Hardy
(Hardy, 1992).
(B,C) The number of hair follicles per unit length (B) and the
percentage of follicles at distinct developmental stages (C) were evaluated
from E18.5 through P1. The number of hair follicles in the mutant was reduced
by approximately 50% relative to WT from E18.5 through P1. At E18.5 in the WT,
primary follicles have developed to stage 3 or 4, whereas secondary follicles
have reached stage 1-2. In the mutant, a reduction was detected from stage 3a
onwards. By P1 in the WT, primary, secondary and tertiary follicles have
developed to stages 5-6, 3-4, and 1-2, respectively. However, by P1 in the
mutant, follicles at stage 1 and after stage 3c were severely reduced relative
to WT.

Effects of Mi-2β deletion on follicular gene expression

The general reduction in hair follicle initiation and development was
confirmed by semi-quantitative RT-PCR. Transcripts of genes preferentially
expressed in the hair follicle epithelium - Edar, β-catenin,
Lef1, Shh, Patched1 and Bmp2 - were consistently reduced in the
mutant skin (see Fig. S2A in the supplementary material). Furthermore, no
increase in the normally low levels of Mi-2α expression was detected in
the mutant skin by either RT-PCR or in situ hybridization (see Fig. S2 in the
supplementary material). Augmented expression of this closely related gene
does not ameliorate the consequences of deleting Mi-2β in embryonic
skin.

Examination of marker gene expression on tissue sections revealed more
dramatic defects in follicle formation. At E18.5 and P1, Edar was detected at
low levels in the basal layer prior to placode formation and expressed more
highly in the developing hair placode. This higher level of expression
persisted in cells at the leading edge of the hair peg as it invaded the
dermis, whereas cells in the rest of the peg exhibited a lower level similar
to that in the interfollicular epidermis
(Fig. 6A,B, WT). At E18.5, many
stage-0 and stage-1 follicles exhibiting bright Edar expression were observed
in WT skin (Fig. 6A,B). By
contrast, although the levels of Edar in the basal epidermis were similar to
those in the WT, no patterned expression of Edar indicative of the initiation
of hair placodes was seen in epidermal regions lacking Mi-2β
(Fig. 6A, mutant). Analysis of
other early molecular markers of follicle induction, including the
downregulation of E-cadherin, induction of P-cadherin, or expression of
Bmp2 or Shh, confirmed the absence of stage-0 or stage-1
follicles in the Mi-2β-depleted regions
(Fig. 6A mutant, and data not
shown). More mature follicles at stage 2-3 of development were observed in
regions lacking Mi-2β, but these were assumed to be tylotricht follicles
that initiated prior to depletion of Mi-2β.

In areas of mosaic Mi-2β depletion, nascent follicular structures
expressing Edar were seen (Fig.
6B,Cb). In these incipient follicles, most of the cells of the
placode expressed Mi-2β but no nascent follicles were forming in adjacent
regions completely devoid of Mi-2β expression. In early follicles with
mosaic Mi-2β depletion, Shh was readily detected at the growing tip of
the follicular epithelium (Fig.
6Ce), whereas in early follicles without any Mi-2β, Shh was
greatly reduced (Fig. 6Cd). By
contrast, in more mature follicles (stage 2/3 or 3a), expression of Shh (and
Edar) was observed, albeit at lower levels, even when Mi-2β was
completely absent (Fig. 6Db and
data not shown). This argues that although Mi-2β activity is required for
the induction of genes involved in follicular morphogenesis, it is not
required for maintenance of their patterned expression at later stages of
development.

The growth of a follicle is dependent on the continued inductive
interactions between DP and the follicular epithelium. Wnt5a was examined as a
marker of the DP that is dependent on expression and signaling of Shh in the
follicular epithelium. Significantly, a marked reduction in Wnt5a expression
was seen in the DP of Mi-2β-depleted follicles, whereas normal levels of
Wnt5a were associated with Mi-2β-expressing follicles in the same animal
(Fig. 6Dd,De).

Taken together, these observations indicate that Mi-2β is required for
the initial patterning of the expression of signaling molecules involved in
follicular morphogenesis. Once committed to a follicular fate, epidermal cells
lacking Mi2β can sustain some follicular development. However, inductive
signaling to and from the DP is impaired and follicular development is
ultimately arrested.

DISCUSSION

Here, we examine the role of the ATP-dependent chromatin remodeler
Mi-2β in the development of skin and its appendages. Our experimental
approach results in loss of Mi-2β in keratinocytes of the developing
integument over the period ranging from before overt epidermal differentiation
through to late gestation. Although Mi-2β depletion occurs over a
developmental continuum, the phenotypes observed suggest that three discrete
steps in the development of the epidermis and its appendages are critically
dependent on Mi-2β activity (Fig.
7). When Mi-2β is depleted during the early commitment of an
ectodermal progenitor to the epidermal lineage in the ventral regions of the
mouse, no immediate effect is observed. The initial differentiation of the
epidermis, several days later, occurs normally. However, epidermal development
is not sustained, suggesting the capacity of the basal epidermis to self renew
is compromised. When Mi2β is deleted in the dorsal epidermis at a later
stage, after a differentiated basal epidermal cell is established, epidermal
differentiation is largely normal and is sustained throughout embryogenesis
and the perinatal period. However, the conversion of a basal epidermal cell to
a progenitor of the pilosebaceous unit does not occur. Finally, when
follicular anlagen are established in the presence of Mi-2β, they can
continue to develop in its absence. However, depletion of Mi-2β after
follicular specification results in arrest of follicular development at stage
3, a period when follicular progenitors are transformed to progenitors of the
hair shaft and inner root sheath in the forming hair bulb.

. Effects of Mi-2β depletion on the signaling network that
controls hair follicle morphogenesis. (A-D) Immunofluorescence of
E18.5 dorsal skin labeled with antibodies against Edar or Edar and E-cadherin
(A), or against Mi-2β and Edar (B,C), or against Mi-2β and
E-cadherin (D). (C,D) In situ hybridization of E18.5 dorsal skin with probes
against Shh and Wnt5a. The depletion of Mi-2β in follicles was confirmed
by immunofluoresence staining of adjacent serial sections using antibodies to
Mi-2β and E-cadherin. DAPI-stained nuclei are blue. (A,B) In the WT, a
local increase in Edar expression was detected among basal epithelial cells
(asterisks) that give rise to the hair placode, as well as within the hair
follicle (arrowhead). Edar upregulation was followed by a decrease in
E-cadherin expression (A, WT). In the mutant, no Edar upregulation or
E-cadherin downregulation was seen in areas of the mutant skin in which
Mi-2β was absent (A, mutant). By contrast, in areas with mosaic
Mi-2β depletion, follicular structures expressing Edar were detected in
the Mi-2β mosaic area in the mutant (arrowhead in B mutant, Cb). Shh
expression was seen at the tip of stage-2 and stage-3a follicles in the WT (Cc
and Da). In the mutant, Shh transcript was seen in stage-2 follicles with
mosaic Mi-2β depletion (Cd), but was significantly reduced in the
Mi-2β-null counterparts (Ce). By contrast, Shh was seen in
Mi-2β-null stage-3a follicles (Db). Expression of Wnt5a was observed in
the dermal condensate of stage-3a follicles in the WT and in
Mi-2β-positive stage-3a follicles in the mutant (Dc and De). Wnt5a was,
however, significantly reduced in the Mi-2β-null counterparts (Dd).

The most severe phenotype associated with early deletion of Mi-2β is
observed in ventral epidermis, whereas the phenotypes associated with later
deletion of Mi-2β predominate in dorsal epidermis. It is formally
possible that the difference in phenotype reflects a fundamental difference in
the physiology of the keratinocytes of the dorsal and ventral compartments,
rather than the timing of Mi-2β deletion. However, the timing of deletion
in the ventral region is also somewhat variable. Patches of epidermis
exhibiting the intermediate phenotype of relatively normal epidermis but
lacking hair follicles, are also observed on the ventrum of some embryos. We
interpret these to be regions where Mi-2β is deleted later in the ventral
region, and conclude that although the different phenotypes observed on the
dorsum and ventrum may be influenced in part by differences in these
keratinocyte populations, the timing of deletion appears to play a more
central role in the phenotype observed. However, the timing of the deletion in
the patches that give rise to the `dorsal' phenotype in ventral epidermis has
not been directly assessed, and a more significant role for differences in the
physiology of dorsal and ventral keratinocytes in the observed differences in
phenotype remains a possibility.

The role of Mi-2β in skin development: a model for the
development of the skin and its appendages and the role of Mi-2β
in this process. Successive stages in the development of wild-type skin
are depicted on the upper time line. We propose that ectodermal cells (yellow)
are first committed to an epidermal TA cell (blue) that can make epidermis but
has limited proliferative potential and developmental plasticity. This cell
type is then converted to an epidermal stem cell (green) with extensive
proliferative capacity and plasticity to adopt alternative fates. Starting at
E14.5, some of these cells are induced to become follicular progenitor cells
(pink), and sometime thereafter other epidermal stem cells give rise to TA
cells of the epidermis with more restricted proliferative and developmental
potential. Follicular progenitors proliferate to make the hair peg, while
epidermal stem and TA cells generate the stratified epidermis. Finally, a
subset of follicular progenitors at the base of the follicle are specified as
the matrix stem cells (orange) that give rise to the hair shaft and inner root
sheath over the anagen phase of the hair cycle (right). These matrix stem
cells are distinct from the follicular bulge stem cells (purple) that
regenerate the lower follicle in the adult. The three phenotypes resulting
from deletion of Mi-2β at different stages of development are shown (1-3,
indicated by a red cross) and interpretations of these phenotypes in the
context of the model are shown beneath.

Distinct transitions in the differentiation of a hair follicle are
dependent on Mi-2β

Of the three transitions in keratinocyte behavior that reveal a crucial
requirement for Mi-2β, only the formation of the epidermal placode of the
hair follicle represents an empirically defined transition in developmental
fate. The follicular epithelium is a lineage compartment that segregates from
the surrounding epidermis at the time of epidermal placode formation
(Levy et al., 2005). In the
dorsal epidermis, Mi-2β is progressively depleted from E14 through birth,
when hair follicles normally form in successive waves. The general reduction
in hair follicle number, and the more specific perturbation of gene expression
and structure within the follicles that do form, must be interpreted in the
context of this mosaic and progressive depletion. Newly initiating placodes
were not observed in the Mi-2β-depleted regions at the early and later
stages of embryogenesis examined in this study. The absence of locally
increased Edar expression, of decreased expression of E-cadherin, or of
induction of Shh expression in these regions, demonstrates that placode
induction is blocked at the very first steps of this process. The fact that
isolated groups of cells expressing Mi-2β within a mosaically deleted
epithelium can form epidermal placodes demonstrates that the development of
inductive signals in the dermis is not impaired, and that Mi-2β is
required in the keratinocytes that must respond to these inductive signals.
Although Mi-2β-deficient basal cells cannot initiate follicle formation,
they can nonetheless sustain the development and differentiation of the
epidermis. Thus, the apparent defect in the basal mutant epidermis is in the
plasticity of these cells to assume the pilosebaceous (follicular) fate
(Fig. 7, phenotype 2).

Although Mi-2β is required for the activation of a battery of markers
in the context of follicle initiation, it does not seem to be directly
required for the maintenance of gene expression patterns once they have been
established. Edar, Shh, Bmp2 and β-catenin are all found in cells lacking
Mi-2β in follicles that presumably formed and activated gene expression
in its presence. In a similar fashion, the suppression of E-cadherin
expression is stable in the absence of Mi-2β. Although the levels of Shh
expression are apparently decreased in less mature follicles lacking
Mi-2β, this is likely to be an indirect effect of a failure in inductive
signaling. A more consistent and presumably previous decline in Wnt5a
expression despite normal levels of Mi-2β in the DP of
Mi-2β-depleted follicles suggests that inductive signaling to the papilla
is compromised in the mutant follicles. This might in turn reduce the levels
of expression of genes in the follicular epithelium that are dependent on
inductive signaling from the DP. The principal exception to the observed
maintenance of gene expression in the absence of Mi-2β is the behavior of
P-cadherin, which appears to decline rapidly in Mi-2β-depleted cells
(data not shown).

During follicle neogenesis, the follicular bulge stem cells arise from
within the follicular epithelium (Levy et
al., 2005), but the timing of this event remains unknown. In a
similar fashion, the segregation of transient matrix stem cells from the cells
that will constitute the permanent portions of the follicular epithelium
during this first hair cycle, is ill defined. It is assumed to occur during
stage 3, when differentiated cell types begin to appear within the follicular
epithelium (Hardy, 1992).
During this stage, the hair matrix is generated and the follicle begins the
transition to an organized structure of concentrically arranged,
differentiated cell types. It is thus noteworthy that a preponderance of
Mi-2β-depleted follicles is arrested in mid-stage 3. As observed in the
initial formation of the epidermal placode, Mi-2β appears to be
preferentially required during the specification of a progenitor population
with a characteristic developmental potential, rather than for the expansion
of cell populations with common developmental potential. Finally, follicles
lacking Mi-2β at later stages of development are observed. These
comparatively rare follicles are likely to represent those that completed the
establishment of the hair matrix stem cells before Mi-2β was
depleted.

Determination of the self-renewal capacity of epidermal precursors by
Mi-2β

Skin development begins from a single layer of embryonic ectoderm that
gives rise to a self-renewing epidermis and its appendages. Mi-2β is
highly expressed in the E10.5 ectoderm, when it begins to commit to an
epidermal/appendage lineage. This suggests that the capacity of Mi-2β to
modify chromatin might be actively required to reprogram the cell fate of
these early progenitors. Nonetheless, depletion of Mi-2β in the E10.5
ectoderm does not interfere with the initial differentiation of the epidermis
that begins a few days later. The differentiation of the successive layers of
the epidermis occurs on schedule. Instead, Mi-2β depletion at this early
stage in skin development appears to alter the properties of the emerging
epidermal precursors allowing them an apparently reduced capacity for
self-renewal and maintenance of the differentiated cell types of the
epidermis. The progressive depletion of squamous layers observed in the
ventral part of the skin, where Mi-2β is depleted early, is consistent
with a defect in the ability of a basal epidermal precursor to regenerate
itself (Fig. 7, phenotype 1).
This effect is unlikely to be due to a defect in the general ability of basal
keratinocytes to enter the cell cycle as normal numbers of proliferating cells
are seen in the basal layer early in development. Reduced proliferation is
only observed after depletion of the basal, suprabasal and granular layers has
begun. Similarly, increased cell death does not account for depletion of the
basal cells. Later deletion of Mi-2β (after E13.5) does not interfere
with the development and maintenance of a multilayered epidermis. Once
established, basal cells subsequently deleted for Mi-2β can sustain
epidermal differentiation and expansion throughout fetal development to the
postnatal stage.

In the parlance of stem cells and TA cells, these studies suggest that as
ectodermal progenitors acquire an epidermal/appendage progenitor fate, they
require Mi-2β to achieve the extended proliferative and self-renewal
capacities of an epidermal stem cell. In its absence, they acquire the more
restricted proliferative capacity and generative potential of a
transit-amplifying cell (Fig.
7, phenotype 1).

Epigenetic regulation in the development of epidermal lineages

The specific blocks at follicular lineage specification, and the subsequent
transition of a follicular progenitor to a matrix stem cell, suggest a crucial
role for Mi-2β and its associates in restructuring a chromatin
environment permissive for the gene expression changes required in the
specified path of differentiation. The brahma (Brm) and brahma-related Brg1
(also known as Smarca4 in mouse - Mouse Genome Informatics)
ATP-dependent nucleosome remodelers act in the context of the SWI2/SNF2
complex and have also been deleted in developing epidermis
(Indra et al., 2005). Ablation
of Brg1, or of both Brg1 and Brm, causes
progressively more severe defects in the later terminal differentiation of the
stratum corneum and in its barrier function. However, defects in earlier
stages of epidermal differentiation or follicular development were not
observed. Thus, the requirement for Mi-2β and possibly the NURD complex
to mediate chromatin remodeling during early differentiation of the integument
is distinct from the functions of the SWI/SNF complex.

Perhaps less clear is how Mi-2β activity might be required to instil
the self-renewal capacity that is lost upon early deletion during epidermal
development. The phenotype of early depletion of Mi-2β is, in some
respects, similar to disruption of p63 activity
(Mills et al., 1999;
Yang et al., 1999). Distinct
isoforms of p63 are thought to be required for the commitment to formation of
stratified epidermis and subsequent maintenance of the proliferative potential
of basal keratinocytes (Koster et al.,
2004; Koster et al.,
2005; Koster and Roop,
2004; McKeon,
2004; Suh et al.,
2006). No gross deregulation of p63 expression is observed in
Mi-2β-depleted skin (data not shown). However, genome-wide analysis of
p63 binding has suggested that chromatin states might regulate p63 access to
cognate sites, and it is possible that Mi-2β activity is permissive for
p63 function (Yang et al.,
2006). Whether mediated by p63 or other factors, a unifying
hypothesis that provides a common mechanistic explanation for the effects of
Mi-2β depletion on the plasticity and self-renewal capacity of
keratinocytes, is that extended self-renewal capacity is actively conferred on
a progenitor with more limited proliferative capacity. In this model, the
self-renewal defects observed in the basal epidermis in ventral skin could be
ascribed to a lack of plasticity in the epidermal progenitor, precluding the
imposition of this aspect of epidermal stem cell character that normally
occurs between E10 and E14 (Fig.
7, phenotype 1).

Defects in terminal differentiation

Although the most dramatic effects of Mi-2β depletion are observed at
critical transitions in cell fate and potential, more modest defects in the
execution of terminal differentiation programs are also detected. Depletion of
Mi-2β in epidermal precursors does not interfere with their ability to
give rise to a multilayered epidermis, as exemplified by the normal expression
of basal and suprabasal layer markers such as K5, K1 and loricrin in the
dorsal side of the mutant skin. However, abnormal expression of K6 was
detected in the suprabasal layers of the mutant animals, but only in cells
that lack Mi-2β. K6 induction occurs in response to defects in terminal
differentiation and/or barrier function of the epidermis. Barrier function
studies indicated a severe defect in the ventral side, but not in the dorsal
side, of the skin where K6 induction is also observed. Although signals
associated with a modestly affected barrier not revealed by the permeability
assay might be responsible for K6 induction, they are only sufficient to
activate K6 in Mi-2β-depleted cells. Whether this reflects a direct
influence of Mi-2β on the keratin gene cluster, or an indirect
consequence of its effects on the physiology of the cell, remains to be
determined.

Conclusion

In summary, the progressive depletion of Mi-2β during the development
of the epidermis in this experimental model has revealed critical transition
points at which this chromatin remodeler is required for the normal
development of the skin and its appendages. Significant changes in the
developmental potential and regenerative capacity of the progenitor cells that
give rise to the epidermis and its appendages depend on the activity of
Mi-2β. Once these changes have been imposed, more modest deficits in the
execution of developmental programs are observed in the absence of Mi-2β.
Further investigation of the mechanisms by which Mi-2β exerts these
effects in this tractable system will provide additional insight into the role
that chromatin remodelers play in the specification of stem cell identity and
potential.

Supplementary material

Acknowledgments

This work was supported by a CBRC Director's Fund and by NIH R01 AI380342
to K.G. We thank Pierre Chambon for providing the K14-Cre mice. We also thank
Bob Czyzewski for mouse husbandry, Janice Brissette and the K.G. laboratory
for discussions and critical comments on the manuscript.